Isothermal Ampilification On Disk Chip Assay For The Detection Of Bacterial Resistance Genes In Lower Respiratory Tractinfections

Objective:In this study,we developed a loop-mediated isothermal amplification(LAMP)assay integrated on a microfluidic disc chip(Isothermal ampilification on disk chip assay),which was capable of simultaneously detecting 20 resistance genes,and the clinical sensitivity and specificity of this assay in batch of bacterial drug resistance gene detection were evaluated by comparison with conventional bacterial culture and drug susceptibility test.Methods:Collecting May-October of 2017,the pactients of Surgical Intensive Care Unit of our hospital,with lower respiratory tract secretions,a total of 103 cases,sent for bacterial culture and bacteria susceptibility test(as the gold standard),at the same time with isothermal ampilification on disk chip assay to detect the samples of 20 drug resistance genes.According to the individual cases,a single gene and the methods of combination were analyzed.Results:1.103 samples through bacterial culture test,49 of germiculture positive,positive rate of 47.6%,produce 10 species,a total of 50 strains of bacterias.they are:baumannii(14 strains),e.coli(6 strains),klebsiella pneumoniae(1strain),klebsiella pneumoniae pneumonia subspecies(9 strains),Streptococcus pneumoniae(1strain),Staphylococcus epidermidis(1 strains),Staphylococcus aureus(6 strains),hemophilies influenza(1 strains),Pseudomonas aeruginosa(11 strains).In addition,there are 4 casesof Blastomyces albicans,and 50 cases were negative.2.The drug susceptibility test was carried out on the 50 pathogenic bacterias,and the results showed that the occurrence frequency of 11 drugs,such as amtrazine and cipropanax,was higher than 20%.3.In all cases,90 cases the gene detection positive,the positive rate of isothermal ampilification on disk chip assay was 87.4%,detected 13 kinds of resistant gene,the detection rate of the top four is erm B,mec A,aad A1 and OXA-66,detection rate of 70.9%,32.0%,29.1% and 32.0%.4.Compared the two kinds of test results,According to each case,the agreement rate was 70.7%(41/58);Calculated according to each resistance genes,the positive rate of isothermal ampilification on disk chip assay was 69%(69/100),the top five gene Mec A(13/16)and OXA-66(12/15),CTX-23(9/11),CTX-M9(5/6)and KPC-2(4/5),the agreement rates were over 80%.Grouped by the category of the resistance genes,resistance gene in carbapenem-Resistant(beta lactamase)OXA-23,OXA-66 and KPC-2,of 2 and 3 genes,the agreement rate were 71.4% and 100%(1 gene agreement rate is 42.9%,);The conformance rate of the two genes of the extended spectrum beta-lactamases(ESBLs)containing the drug-resistant gene CTX-M1 and CTX-M9 was 100%(the agreement rate of one gene was 85.7%).Conclusion:13 kinds of drug-resistant genes can be detected by isothermal ampilification on disk chip assay in one time,and the detecting speed and the positive rate were significantly higher than that of traditional bacterial culture and drug susceptibility test.The agreement rate of this two methods is good.Especially,the six genes Mec A,OXA-23,KPC-2,OXA-66,CTX-M1 and CTX-M9,in the form of a single gene or combination,the test results has a higher agreement rate.So,this assay has a good prospect in clinical application,and is worthy of further research and popularization.